Experimental Hematology 28 (2000) 1451–1459
Autonomous behavior of hematopoietic stem cells
Leonie M. Kammingaa, Imre Akkermana, Ellen Weersinga,
Albertina Ausemaa, Bert Dontjea, Gary Van Zantb,c, and Gerald de Haana
a
Department of Stem Cell Biology, University of Groningen, Groningen, The Netherlands;
Division of Hematology/Oncology, Blood and Marrow Transplant Program, Lucille P. Markey
Cancer Center, Lexington, Ky., USA; cDepartment of Physiology, University of Kentucky Medical Center, Lexington, Ky., USA
b
(Received 19 May 2000; revised 24 July 2000; accepted 27 July 2000)
Objective. Mechanisms that affect the function of primitive hematopoietic stem cells with
long-term proliferative potential remain largely unknown. Here we assessed whether properties of stem cells are cell-extrinsically or cell-autonomously regulated.
Materials and Methods. We developed a model in which two genetically and phenotypically
distinct stem cell populations coexist in a single animal. Chimeric mice were produced by
transplanting irradiated B6D2F1 (BDF1) recipients with mixtures of DBA/2 (D2) and C57BL/
6 (B6) day-14 fetal liver cells.
Results. We determined the mobilization potential, proliferation, and frequency of D2 and B6
stem and progenitor cells in animals with chimeric hematopoiesis. After granulocyte colonystimulating factor (G-CSF) administration, peripheral blood D2 colony-forming units granulocyte-macrophage were fourfold to eightfold more numerous than B6 progenitors. We determined that D2 and B6 progenitors maintained their genotype-specific cycling activity in BDF1
recipients. Chimeric marrow was harvested and D2 and B6 cell populations were separated by
flow cytometry. Cobblestone area-forming cell (CAFC) analysis of sorted marrow showed that
the number of late appearing CAFC subsets within the D2 cell population was zthreefold
higher than within the B6 fraction. We performed secondary transplantation using unfractionated chimeric marrow, which was given in limiting doses to lethally irradiated BDF1 recipients. Comparison of the proportion of animals possessing D2 and/or B6 leukocytes 5
months after transplant revealed that the frequency of D2 LTRA was z10-fold higher than B6
LTRA numbers.
Conclusion. Our data demonstrate that genetically distinct stem cell populations, coexisting in
individual animals, independently maintain their parental phenotypes, indicating that stem
cell properties are predominantly regulated cell-autonomously. © 2000 International Society
for Experimental Hematology. Published by Elsevier Science Inc.
Keywords: Stem cells—Genetics—Transplantation—Fetal liver—Chimerism
Introduction
Peripheral blood cell numbers are homeostatically maintained within a narrow range to ensure that essential physiological processes are carried out effectively. Studies of mice
bearing mutant genes that encode specific lineage-restricted
growth factors or receptors have conclusively demonstrated
that the pool size of mature blood cell subsets is regulated
by extrinsically acting hematopoietic growth factors [1–3].
Whereas the number of circulating peripheral blood cells is
Offprint requests to: G. de Haan, Ph.D., Department of Cell Biology,
University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The
Netherlands; E-mail: g.de.haan@med.rug.nl
similar among normal healthy humans and in most inbred
strains of mice, the frequency of primitive hematopoietic
stem cells varies widely [4–7]. This suggests that regulatory
mechanisms affecting the frequency of stem cells in the
bone marrow are distinct, and at least partly independent,
from those that maintain normal levels of mature cells in the
blood. Given the expanding diversity of potential clinical
applications for which hematopoietic stem cells, and indeed
also stem cells derived from other somatic tissues [8–10],
can be of use to the patient, the issue as to how stem cell
identity is regulated is of fundamental importance. Pertinent
to the question of which roles stem cells may play in clinical
applications is the extent to which these cells can be manipulated extrinsically, either in vivo or in vitro.
0301-472X/00 $–see front matter. Copyright © 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.
PII S0301-472X(00)0 0 5 4 3 - 9
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L.M. Kamminga et al./Experimental Hematology 28 (2000) 1451–1459
Conceptually, stem cell homeostasis could be achieved
by cell-extrinsic or cell-intrinsic mechanisms, and these two
pathways need not be mutually exclusive. To address this
issue, we studied qualitative and quantitative competitive
repopulation potential of genetically distinct stem cell populations, coexisting in a common environment in vivo. This
was accomplished by transplanting stem cells obtained from
fetal livers of gestational day 14 D2 and B6 embryos into lethally irradiated BDF1 recipients. To avoid “graft-vs-graft”
reactions, we elected to use fetal liver, instead of adult bone
marrow, as a source of stem cells. At this stage of ontogeny,
no mature T and NK cells are present in the fetal liver [11],
rendering both genetically distinct grafts immunologically
compatible. We and others have shown that D2 mice have a
large stem cell pool with actively cycling progenitors,
whereas B6 mice have fewer stem cells with slowly proliferating progenitors [4,12–14]. In addition, possibly as a
consequence of distinct stem cell pool sizes, D2 progenitor
cells are much more readily mobilized to the peripheral
blood after G-CSF administration than B6 cells [5,15]. Using a variety of in vitro and in vivo techniques, we show that
the two stem cell populations maintain their parental phenotype within the common environment of the chimera. These
data support the notion that normal hematopoietic stem
cells, not unlike their malignant counterparts [16], autonomously regulate their own cell number.
Materials and methods
Mice
Female B6D2F1OlaHsD and timed pregnant C57BL/6JOlaHsD
and DBA/2OlaHsD mice were purchased from Harlan, Horst, The
Netherlands. BDF1 mice were used as recipients at an age of 10–
12 weeks.
Production of fetal liver chimeras
BDF1 mice were lethally irradiated with 9.5 Gy using an IBL 637
Cesium-137 g source (CIS Biointernational, Gif-sur-Yvette,
France), 24 hours prior to transplantation. Pregnant B6 and D2
mice (14 days postcoitus) were sacrificed by cervical dislocation
and fetal livers were isolated. A single-cell suspension was prepared by repeated flushing through increasingly thinner needles
and cell yield was counted using a Coulter Counter (Coulter Electronics, LTD, Dunstable, England). BDF1 mice were transplanted
with either D2 cells alone (n 5 4), B6 cells alone (n 5 4), or a 1:4
mixture of D2 and B6 cells respectively (n 5 3). All mice received
a total of 2 3 106 cells, injected intravenously into the retro-orbital
plexus. Fourfold more fetal liver cells from B6 than D2 were transplanted since we expected D2 fetal liver cells to have superior repopulating potential compared to B6 cells, as the frequency of
primitive stem cell subsets in the fetal liver of D2 mice is significantly higher [17].
Determination of peripheral blood
cell chimerism using flow cytometry
At various times after transplantation, approximately 50 ml blood
was drawn from the retro-orbital plexus of each mouse and erythro-
cytes were lysed by hypotonic shock (0.16 M NH4Cl, 1.0 3 1024
M EDTA, 0.017 M NaCl, 5 minutes at room temperature). Leukocytes were washed and stained with fluorescein isothiocyanate
(FITC)-labeled anti H-2Kd-antibody (Pharmingen, San Diego, CA),
and biotinylated anti H-2Kb (clone B8-24-3, a gift from Dr. S.J. Szilvassy) cells were incubated for 40 minutes at 48C, washed twice,
and subsequently incubated an additional 40 minutes at 48C with
Streptavidin-phycoerythrin (Str-PE) (Pharmingen, San Diego, CA).
After washing, cells were finally resuspended in 200 ml phosphatebuffered saline (PBS) 1 2% fetal calf serum (FCS). Two-color
analysis (FACSCalibur, Becton-Dickinson, Palo Alto, CA) was
performed to assess the percentages of leukocytes derived from D2,
B6, and BDF1. D2 cells are H-2Kd1 whereas B6 cells are H-2Kb1.
BDF1 cells possess both H-2K isotypes, allowing accurate quantification of leukocyte contribution of all three genotypes.
Progenitor mobilization with SD/01
All transplanted mice, including four additional mice of each parental strain, received a single subcutaneous injection of 25 mg
polyethyleneglycol-conjugated recombinant human G-CSF (PEGG-CSF, referred to as Sustained Duration/01, kindly provided by
Amgen Inc., Thousand Oaks, CA) dissolved in 200 ml PBS with
2% FCS. SD/01 has been demonstrated to potently mobilize stem
and progenitor cells from the bone marrow to the blood [18]. Progenitor cells were harvested from the peripheral blood three days
after administration of SD/01, a time point coinciding with peak
increase in progenitor cell numbers in the blood [18].
Hydroxyurea suicide technique
To measure the number of cells in S phase, the fraction of cells
killed by a 1-hour incubation with hydroxyurea (Sigma, St. Louis,
MO, 200 mg/mL) was determined as we have reported before [4].
Bone marrow cells were obtained from anesthesized mice by inserting a 30G needle into the knee joint [19]. 30 ml of medium was
first injected into the marrow cavity and cells were subsequently
aspirated. The average yield of these aspirates was z1 3 106 cells.
Cell suspensions were diluted to a volume of 1 mL, and divided in
two samples. Hydroxyurea was added to one sample, and incubated with the control sample for 1 hour at 378C. Both cell suspensions were then washed, and CFU-GM cultures were initiated. The
fraction of cells killed by hydroxyurea was calculated and was
considered to reflect the percentage of cells in S phase.
CFU-GM culture assay
CFU-GM were determined using standard methylcellulose cultures (0.8% methylcellulose, 30% FCS in alpha medium). Cultures, supplemented with 10 ng/mL recombinant mouse GM-CSF
(Behringwerke, Marburg, Germany) and 100 ng/mL recombinant
rat PEG-SCF (Amgen), were inoculated with 5–15 ml of whole heparinized blood/mL. Colonies were cultured at 378C and 5% CO2
and scored after 6 days.
Genotyping individual CFU-GM colonies
To genotype individual CFU-GM, colonies were picked from the
culture using a micropipet and suspended in 100 ml PBS. Cells
were washed and the pellet was resuspended in 10 ml DNA isolation buffer (50 mM KCl, 1.5 mM MgCl2, 10 mM Tris HCl, pH 8.5,
0.01% gelatin [Sigma, St. Louis, MO], 0.45% Igepal [Sigma],
0.45% Tween 20 [Sigma], and 100 mg/mL proteinase K [GibcoBRL]) to release DNA from the cells. Samples were incubated at
558C for two hours and subsequently at 958C for 15 minutes to in-
L.M. Kamminga et al./Experimental Hematology 28 (2000) 1451–1459
activate proteinase K. Samples were stored at 2208C until use in
PCR. The genotype of each colony was verified using primers
(MapPairsTM, Research Genetics Inc., Huntsville, AL) that amplify
the simple sequence length polymorphism D14Mit266; the D2 allele of this microsatellite is 180 bp and the B6 allele is 148 bp. A
10 ml PCR reaction was performed according to the protocol provided by the manufacturer using a PerkinElmer GeneAmp PCR
System 9700 (PerkinElmer Corp., Norwalk, CT). Amplified DNA
was resolved on a 3,5% ethidium bromide–stained agarose gel.
Sorting of D2 and B6 bone marrow cells from chimeric mice
Twenty weeks after transplantation chimeric mice were sacrificed
and bone marrow cells were isolated from both femurs. After staining
total bone marrow cells with anti–H-2Kb and anti–H-2Kd antibodies
as described, B6 and D2 cell populations were separated using a
MoFlo flow cytometer (Cytomation, Ft. Collins, CO). D2 and B6
cell populations were collected and separate cobblestone areaforming cell (CAFC) assays were performed on both samples.
Cobblestone area-forming cell assay
Stem cell activity was assessed using in vitro limiting dilution type
long-term bone marrow cultures. The cobblestone area-forming
cell assay was performed as described previously [4]. For each of
the six cell dilutions used, 20 replicate wells were tested. Early appearing CAFC day-7 correspond to relatively committed progenitor cells, whereas late appearing CAFC day-35 reflect more primitive cell subsets [20]. CAFC frequencies were calculated using
maximum likelihood analysis [21].
Secondary transplantation
Twenty weeks after the initial fetal liver stem cell transplantation, a
single chimeric mouse was sacrificed by cervical dislocation. Cells
were isolated from both femurs and transplanted in limited dilution
into a new cohort of lethally irradiated BDF1 recipients. Recipients
(5 per group) received 2.0 3 106, 1.0 3 106, 0.5 3 106, or 0.2 3 106
chimera-derived bone marrow cells. Engraftment of B6 and D2 leukocytes was assessed using H-2K phenotyping. Using Poisson statistics the proportion of secondary recipient animals that showed B6
or D2 leukocytes 12 weeks after transplantation was used to calculate the frequency of B6 and D2 long-term repopulating stem cells.
1453
three cell populations derived from B6, D2, and BDF1 stem
cells can easily be distinguished. Figure 2 shows the contribution of stem cells from each genotype to the total leukocyte pool. Panel A depicts chimerism after injection of 2 3
106 D2 fetal liver cells. After 2 months almost all leukocytes
(.95%) originated from donor stem cells, whereas only a
few cells were host-derived. Similarly, leukocyte reconstitution in BDF1 mice injected with 2 3 106 B6 cells was almost entirely of B6 origin (Panel B). Importantly, no differences in either engraftment kinetics or engraftment level
were observed between these two cell sources, demonstrating that there was no preferential rejection by BDF1 recipients of stem cells of either B6 or D2 genotype. Panel C
shows engraftment kinetics in recipients transplanted with
mixed D2/B6 stem cells. Although mice received fourfold
more B6 fetal liver cells, D2 peripheral blood leukocytes
were twofold to threefold more abundant than B6 leukocytes for the first 100 days after transplant. This indicates
that D2 fetal liver cells have an eightfold to 12-fold higher
repopulating potential compared to B6 cells. However, it
should be noted that DBA reconstitution decreased with
time, and B6 leukocytes became more prominent at later
time points after transplant. This has also been observed in
B6 ↔ D2 embryo aggregation chimeras, although in these
animals skewing occurred only after 12–18 months [23].
D2 and B6 progenitor cell cycling in chimeras
We have previously shown that D2 progenitors have a higher
steady state cycling activity than B6 cells [12,13,17]. We isolated bone marrow cells from BDF1 mice transplanted 5
months earlier with only D2 or only B6 fetal liver cells (Fig.
Results
Development of radiation chimeras
Chimeric mice were produced by transplanting D2, B6, or a
mixture of D2 and B6 fetal liver cells in lethally irradiated
BDF1 recipients. To ascertain that genotype-restricted hybrid resistance (i.e., preferential rejection of donor cells of
either parent by the F1 recipient) did not bias our results, we
included two groups of recipients that received only D2
cells or only B6 cells (2 3 106 cells). Chimeric mice were
created by transplanting 0.4 3 106 D2 fetal liver cells,
mixed with 1.6 3 106 B6 fetal liver cells. A 1:4 ratio of D2
vs B6 cells was used because we expected D2 cells to have
a better repopulating ability [22] and because we had previously demonstrated that the frequency of primitive cells in
the D2 fetal liver is zthreefold higher than in the B6 liver
[17]. The dot plot shown in Figure 1 demonstrates that all
Figure 1. Dot plot showing the presence of D2 (H-2Kd1) and B6 (H-2Kb1)
leukocytes in the peripheral blood of lethally irradiated BDF1 mice (H2Kbd1), transplanted with 2.0 3 106 fetal liver cells in a 1:4 D2:B6 ratio.
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L.M. Kamminga et al./Experimental Hematology 28 (2000) 1451–1459
Figure 3. Proliferation of progenitors. The percentage of CFU-GM in S
phase in the bone marrow of lethally irradiated BDF1 recipients transplanted with D2 fetal liver cells only (D2 . F1) or B6 fetal liver cells only
(B6 . F1).
Figure 2. Chimerism in peripheral blood. Panel A shows the percentages
of donor-derived D2 leukocytes in the peripheral blood of lethally irradiated BDF1 mice after transplantation of 2.0 3 106 D2 fetal liver cells. Panels B and C show chimerism after transplantation of 2.0 3 106 B6 fetal
liver cells and 0.4 3 106 D2 1 1.6 3 106 B6 fetal liver cells, respectively.
Values are given 61 SEM.
2A and B) and determined the percentage of CFU-GM in S
phase. Progenitors in mice transplanted with D2 stem cells
had an average percentage of 28% in S phase, whereas only
9% of the progenitors in mice transplanted with B6 cells were
in S phase (Fig. 3). These values were identical to those typically observed in the parental strains [13,17].
Genotype-restricted mobilization
potential of hematopoietic progenitors
In humans and in various inbred strains of mice, the number
of primitive cells mobilized from the bone marrow into the
peripheral blood after growth factor administration varies
widely [5,7,15,24]. We have previously postulated that differential mobilization responses reflect distinct marrow
pool sizes [6]. To test this hypothesis we administrated SD/
01, a pegylated formulation of rhG-CSF with enhanced biological activity [18], to parental and transplanted chimeric
mice. The number of CFU-GM/mL blood, three days after
injection of SD/01 to parental D2 and B6 mice, is shown in
Figure 4A. The large strain difference is apparent and is in
full agreement with previously reported data [5,15]. Mobilization potential of irradiated F1 animals, transplanted with 2
3 106 B6 or 2 3 106 D2 fetal liver cells, is depicted in Panel
B of Figure 4. No significant differences compared to parental strains were observed, indicating that transplantation
and engraftment of donor stem cells does not detrimentally
affect subsequent mobilization potential. Since B6 and D2
progenitor cells maintained their characteristic parental mobilization pattern in an F1 microenvironment, these data additionally demonstrate that genotype-restricted differences
in mobilization response after G-CSF represent an intrinsic
stem cell trait.
Fifteen weeks after transplantation, a time point when
D2-derived leukocytes were ztwofold more frequent than
B6 leukocytes in recipients of mixed B6 and D2 fetal liver
cells (Fig. 2C), SD/01 was administered to the chimeras,
and three days later CFU-GM were cultured from peripheral
blood. Individual CFU-GM colonies were genotyped using
primers that amplify the polymorphic microsatellite marker
D14Mit266. Figure 4C shows an example of a genotyping
experiment in which seven D2, four B6, and one BDF1 colonies were detected. Fifty colonies were analyzed in each
L.M. Kamminga et al./Experimental Hematology 28 (2000) 1451–1459
1455
Figure 4. Progenitor mobilization potential. Number of CFU-GM per mL blood three days after administration of SD/01 to parental strains (Panel A), or to
chimeric mice transplanted with only D2 or only B6 fetal liver cells (Panel B). Mobilized colonies in the peripheral blood of the chimeras were genotyped
using informative SSLP-PCR. Panel C depicts the amplified PCR products (D2 allele is 180 bp and B6 allele is 148 bp). Panel D shows the number and genotype of CFU-GM per mL peripheral blood in three BDF1 recipients that received 0.4 3 106 D2 1 1.6 3 106 B6 fetal liver cells.
chimera. The total number of B6, D2, or BDF1-derived
CFU-GM/mL blood from each chimeric mouse is depicted
in Panel D of Figure 4. Endogenous BDF1-derived colonies
were observed rarely. The frequency of D2-derived CFUGM was fourfold to eightfold higher than B6-derived progenitors. The skewed prevalence of D2 progenitors exceeded
by severalfold the relative frequency of total D2 leukocytes,
suggesting either that D2 progenitor cells were more efficiently mobilized than were B6 cells, or, alternatively, that
the marrow frequency of D2 stem and progenitor cells was
significantly higher than B6 cells.
D2 and B6 stem cell frequencies in
chimeric mice; a limiting dilution in vitro analysis
To assess whether the overrepresentation of D2 progenitors
after mobilization resulted from a higher D2 stem cell frequency in the bone marrow of the chimeras, we measured
the pool size of D2 and B6 stem cells. First, an in vitro cob-
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L.M. Kamminga et al./Experimental Hematology 28 (2000) 1451–1459
blestone area-forming cell (CAFC) assay was performed using FACS-sorted D2 and B6 cell populations obtained from
bone marrow of the chimeras. Importantly, at the time point
of sacrifice (140 days after transplant) B6 and D2 leukocyte
populations in the peripheral blood were equally frequent in
these chimeric mice (Fig. 2C). After staining bone marrow
cells with anti H-2Kd and anti H-2Kb antibodies, D2 and B6
cell fractions were separated using flow cytometry, and independent CAFC assays were performed on both populations.
In the chimeric bone marrow, 56% of the cells were D2derived and 33% were B6-derived (Fig. 5A). CAFC day-7
frequency was twofold higher in the H-2Kd1-cell fraction
compared to cobblestone area-forming activity in the H-2Kb1
cell fraction. (Fig. 5B). For comparison, the CAFC day-7 frequency in unfractionated parental D2 and B6 control bone
marrow is shown as well. CAFC day-35 were threefold more
frequent in the H-2Kd1cell fraction compared to the H-2Kb1
cell fraction. (Fig. 5C and D). These values were similar to
the stem cell frequency in the parental strains (Fig. 5C).
D2 and B6 stem cell frequencies in
chimeric mice; a limiting dilution in vivo analysis
To confirm that the distinct cobblestone area-forming activity of H-2Kd1 and H-2Kb1 cell fractions properly reflected
stem cell activity, and thus to reinforce our in vitro CAFC
data, we performed a limiting dilution long-term repopulating ability (LTRA) assay. To this end, bone marrow was
isolated from a chimeric animal and transplanted without
further manipulation into a secondary cohort of lethally irradiated BDF1 mice in four cell doses (2.0 3 106, 1.0 3 106,
0.5 3 106, or 0.2 3 106 cells). At the time of bone marrow
harvest (18 weeks after transplantation), 45% of the leukocytes were D2 derived and 40% were B6 derived. Figure 6A
shows the percentage of secondary F1 recipients that failed
to show more than 0.5% (detection limit) B6 or D2 lymphocytes or granulocytes 5 months posttransplant, as a function
of the number of cells transplanted. In all four groups, each
consisting of five animals, were mice that did not have B6
leukocytes. Only a single recipient, transplanted with the
lowest cell doses of 0.2 3 106 cells, did not show long-term
D2 granulocyte contribution (chimera 20, Fig. 6B). In contrast to the initial fetal liver chimeras, this cohort of secondary recipients showed considerable variation in levels of
chimerism within each group (Fig. 6B). Using Poisson statistics, the frequency of B6 repopulating stem cells was estimated to be 0.08 per 105 bone marrow cells, whereas the
frequency of D2 repopulating stem cells was 10-fold higher,
0.8 per 105 (Fig. 6C). In good agreement with the in vitro
CAFC data, these in vivo results show that D2 stem cells are
much more frequent than B6 stem cells in the common environment of the F1 chimera.
Discussion
In this study we wished to determine whether specific functional characteristics of hematopoietic progenitor and stem
Figure 5. Cobblestone forming activity in chimeric bone marrow. Percentage D2- and B6-derived bone marrow cells in chimeras, 140 days posttransplant, is
shown in panel A. Panels B and C show the frequency of CAFC day-7 and day-35 respectively, within the H-2Kd1 and H-2Kd1 cell fraction, isolated from the
chimeric bone marrow by flow cytometry. Also shown in panels B and C are values for these cell stages as measured in parental control animals. Panel D
illustrates the presence of cobblestone areas within the H-2Kb1 and H-2Kd1 cell fractions after 35 days of culture as a function of the number of cells inoculated per well.
L.M. Kamminga et al./Experimental Hematology 28 (2000) 1451–1459
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Figure 6. LTRA measurements using chimeric bone marrow graft. Percentage of secondary recipients that failed to show multilineage B6 or D2 contribution
after transplantation of chimeric marrow obtained 5 months after primary fetal liver transplantation is shown in panel A. Panel B depicts the percent B6 and
D2 granulocytes in the peripheral blood of each of the 20 chimeras as a function of transplanted cell dose. Panel C shows the LTRA frequency estimates of the
corresponding limiting dilution analysis.
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L.M. Kamminga et al./Experimental Hematology 28 (2000) 1451–1459
cells are defined cell-autonomously or by signals from the
microenvironment. Cell traits that we analyzed included the
rate of proliferation, the ability to form colonies in longterm stroma-associated cultures, the ability to migrate to the
peripheral blood after cytokine challenge, and, finally, the
ability to reconstitute the hematopoietic system of a lethally
irradiated recipient. We took advantage of the fact that different inbred strains of mice have been shown to display a
widely varying range of phenotypes related to these aspects
of stem cell biology. Compared to B6 mice, D2 mice have
more stem cells [6,14 22] and a higher proportion of progenitor cells in S phase [12,13,25], which are more easily
mobilized to the peripheral blood [5,15].
Using a competitive analysis in which D2 hematopoietic
stem cells derived from the fetal liver were allowed to compete with similarly obtained B6 stem cells, we show conclusively that all strain-specific characteristics were maintained with high fidelity within the common environment of
the chimeras. Thus, all data presented in this study argue
strongly for an intrinsic modulation of stem cell function.
One of us (G. Van Zant) has reported extensively on hematopoietic chimerism in B6 ↔ D2 embryo aggregation
chimeras [23,26]. Stem cell proliferation, stem cell frequency, and stem cell mobilization were never directly measured in those chimeras. However, when bone marrow cells
obtained from these chimeras were transplanted into lethally irradiated F1 animals, the large majority of leukocytes were derived from D2 stem cells, very similar to what
we have observed in the current study using fetal liver radiation chimeras. This competitive advantage was considered
to be a consequence of the higher proliferative fraction of
D2 cells compared to B6 cells. Although differential cell
proliferation probably contributes to this process, our
present studies show in fact that D2 stem cells are substantially more abundant than B6 stem cells in the chimeras.
Our current results are in good agreement with those recently published by Muller-Sieburg et al., who also used the
above mentioned embryo-aggregation technique. LTC-IC
measurements in chimeric mice revealed consistently a
higher frequency of D2 stem cells than expected based upon
chimerism in other somatic tissues [27].
We have recently shown that the in vitro proliferative potential of a highly enriched single Lin2Sca-11 c-kit1 D2
stem cell is substantially higher compared to its B6 counterpart [22]. Although our present data at first sight also suggest that D2 stem cells are “superior” compared to B6 cells,
production of mature cells by D2 stem cells is not very efficient in the chimeras, since a large D2 stem cell compartment produces an equal number of leukocytes as a small B6
compartment. This paradox is also observed in parental
mice; in B6 mice few stem cells and few actively cycling
progenitors maintain normal blood cell values as efficiently
as D2 mice with many stem cells and highly cycling progenitors. This suggests that down-stream mechanisms, such
as distinct rates of apoptosis of committed cells or the num-
ber of amplification divisions, may further regulate peripheral blood cell production.
The extent to which stem cell population size in vivo is
affected by cues from the systemic or local environment potentially predicts limitations to ex vivo expansion protocols.
Attempts to multiply and manipulate hematopoietic stem
cells are currently being explored in many laboratories. If
stem cell function is predominantly intrinsically regulated,
as our data imply, it may prove difficult to expand these
cells in vitro using exogenously added growth factors. It
may therefore be significant that hematopoietic stem cells
have been notoriously difficult to maintain in culture.
Rather, altering the intrinsic genetic program of stem cells
may prove to be more fruitful. First examples of genetic
(re)programming are provided by studies showing that stem
cells overexpressing HoxB4 or bcl-2 have enhanced repopulating activity, whereas cells deficient of the cyclin-dependent kinase inhibitor p21 have reduced stem cell activity
[28–30]
Recently it has been argued that cell-autonomous behavior is a property of malignantly transformed leukemic cells
[16]. Our data indicate that normal primitive hematopoietic
stem cells also display autonomous characteristics. At
present, the molecular nature of such stem cell autonomy
has yet to be defined. Truly intrinsic effectors, such as differentially expressed transcription factors, may play a role.
Alternatively, however, secreted glycoproteins expressed
by stem cells may affect stem cell pool size in an autocrine
fashion. We, and others, have previously mapped chromosomal regions associated with variation in stem cell frequency and mobilization potential [6,13–15,31]. Although
the underlying genes have not yet been identified, our current experiments indicate that these genes must be expressed by the stem cells themselves.
Acknowledgments
G. deHaan is a fellow of the Netherlands Organization for Scientific
Research (NWO) and of the Royal Netherlands Academy of Arts
and Sciences (KNAW). This work was supported by funds provided
by NIH grant RO1 AG16653 to G. Van Zant, and the Lucille P.
Markey Cancer Center, the University of Kentucky Hospital, and
the Department of Internal Medicine. The authors would like to
thank Geert Mesander for excellent assistance with flow cytometry
and Dr. Piet Wierenga for intravenous injection of fetal liver cells.
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